ABSTRACT Heme proteins are fundamental in biology yet we do not understand the mechanisms that regulate their maturation and function in cells, or how they become dysfunctional in disease. Our lab is discovering new roles for cell chaperones and nitric oxide (NO) in regulating these processes. Soluble guanylate cyclase (sGC), myoglobin (Mb), and NO synthase (NOS) will serve as the model heme proteins in our proposal. sGC becomes dysfunctional in inflammatory diseases with higher NO production. Our work reveals that cells chaperones (hsp90) govern the maturation of all three hemeproteins, but the mechanisms driving their heme insertion, and in the case of sGC, that drive its inactivation, are unclear. We hypothesize: (i) In health, hsp90 supports maturation of sGC, Mb, and NOS through making a direct interaction with each client protein that enables their heme insertion, likely operating in concert with co-chaperone proteins. (ii) In disease, higher NO inactivates sGC by causing oxidation of its heme and S-nitrosation of its protein Cys groups (SNO). The SNO modifications in turn causes sGC heterodimer breakup, possible heme loss from sGC?, and sGC? re-association with hsp90. (iii) Cell denitrosylase (thioredoxin 1, Trx1) and sGC heme reductase (cytochrome b5r) enzymes may protect and/or repair sGC. To understand the molecular basis for these events, our Aims coordinate biochemical, biophysical, & cell-based approaches. AIM 1. How do chaperones drive heme insertion? Determine regions in apo-Mb and apo-NOS that enable complex formation with hsp90, build structural model of the complexes, test if interaction as predicted by models enable complex formation and heme insertion to occur in mammalian cells. Identify the co-chaperones and proteins that may assist hsp90 during heme insertions into the sGC?, Mb, and NOS clients. Create defined protein systems to study their heme insertion. Characterize structure of hsp90-apo-hemeprotein client complexes by HxD MS and EM. AIM 2. How is sGC inactivated & how might its recovery take place? In cells and in a purified system, determine the importance of: (i) sGC? heme occupancy & heme oxidation state in catalyzing specific SNO in modifications in sGC?/?, (ii) SNO modifications vs heme oxidation or loss in driving sGC heterodimer breakup & the rebinding of sGC? to hsp90; (iii) protective/recovery mechanisms, including SNO removal by Trx1, hsp90- mediated heme reinsertion, sGC? heme reduction by cytochrome b5r, & binding of a drug that occupies the sGC? heme site (BAY 60). By defining the molecular & cellular mechanisms of chaperone-driven heme insertion during sGC, Mb, and NOS maturation, and the mechanisms causing sGC inactivation and recovery, our study will make fundamental contributions to our understanding of hemeprotein maturation and function, and will reveal new ways to optimize hemeprotein functions in health and disease.